We have gathered strong evidence that the synaptotoxicity of prions depends on activation of specific signal transduction pathways that cause changes in protein phosphorylation mediated by key protein kinases, and that result in downstream alterations in gene transcription. In this project, we propose to use cutting-edge proteomic techniques and RNA-Seq to analyze global changes in protein phosphorylation and gene transcription in neurons undergoing the earliest detectable changes in synaptic structure and function. We will use specialized cultures of hippocampal neurons that respond rapidly (within hours) to neurotoxic forms of PrPSc by retraction of dendritic spines and by alterations in synaptic transmission. We then plan to process these data using sophisticated bioinformatics pipelines to identify cellular signaling pathways and transcriptional networks mediating the synaptotoxic effects of PrPSc. We anticipate that the comprehensive, discovery-based approach outlined here will yield important clues to the underlying biology of prion diseases, and will identify a wealth of novel therapeutic targets for ameliorating synaptic degeneration in these disorders.